Cross-linked cellulose sponge

ABSTRACT

A cross-linked flexible sponge absorbent medium containing substantially uniformly distributed fibrous reinforcement is prepared under such conditions that swelling of the sponge is controlled to a water retention value of 2 to 6; and simultaneously or subsequently, the cross-linked sponge is reacted with a reagent which introduces functional groups into the modified cross-linked sponge. There is also provided apparatus having an inlet for liquid, an outlet for liquid so as to define a path for liquid flow between the inlet and the outlet, an adsorbent medium, obtainable by the above method, being positioned across the liquid flow path in the apparatus.

This application is a division of U.S. patent application Ser. No.07/945,652, filed on Nov. 2, 1992, now U.S. Pat. No. 5,492,723.

The present invention is concerned with sponge adsorbent media havingfunctional groups chemically bonded thereto.

There are two broad ways of making cellulosic materials in the form offlexible sponge and having functional groups (such as ion-exchangegroups) chemically bonded to the cellulose. In one method, as describedin GB 914421, a pre-formed flexible cellulosic sponge is modified byreaction with a reagent which introduces ion-exchange groups (such asorthophosphoric acid or sodium chloroacetate). GB 1387265 disclosesion-exchange cellulosic material prepared by reaction of cellulose witha reagent which introduces ion-exchange groups, followed by regenerationinto the desired physical form, among which is sponge.

GB 1226448 discloses a method of making an ion exchanger, whichcomprises the introduction of cross-linking residues into regeneratedcellulose together with or followed by introduction of cation or anionexchange groups into the cellulose. The cellulose is typically obtainedfrom viscose; no preferred physical form for the cellulose is specified(i.e. the cellulose may be used in a variety of physical forms such asrod, filament, yarn, woven cloth, flakes, beads, granules, powder,sponge, tube or sheet).

The present invention is concerned with a method of preparing spongeadsorbent media having a pre-determined porous structure and whichmethod involves modification of a flexible sponge.

Hydrophilic cellulosic chromatographic media have been widely used forthe isolation or separation of macromolecules, such as proteins, both inthe laboratory and on a commercial scale. However, sponge adsorbentmedia have not been used greatly in commercial chemical separationoperations (such as ion-exchange separation techniques), probablybecause of the absence of any sponge adsorbent media possessing therequired porous structure and the resulting difficulties of ensuringadequate contact between the liquid being treated and the sponge. Forthis reason, particulate and granular media are generally used, despitethe disadvantages associated with the use thereof such as slow flowrates, plugging of the bed, and mal-distribution of flow and also theneed for considerable skill in filling a column to avoid channelling.

The mass transfer rate of substances being treated by means of adsorbentmedia is generally diffusion limited. The adsorption reaction at thesurface of adsorbent media is fast, whilst limiting processes foradsorption and elution are the film diffusion resistance around thematrix and the pore diffusion resistance within it. Higher flow rateswill reduce the resistance of film diffusion and hence increaseproductivity by decreasing process time. The resolution of ion-exchangechromatography requires the column to have a certain length, the flowrate being inversely proportional to column length. When recovering abiological molecule present in very low concentration in a large volumeof feedstream, flow rate and capture efficiency are the major factors tobe optimised.

We have now devised a method of producing cellulosic sponge adsorbentmedia having superior flow properties and adsorption and desorptionkinetics, and which are also specifically modified so as to be suitablefor use as chromatographic adsorbents.

According to the invention, there is provided a method of preparing asponge adsorbent medium, which method comprises:

preparing a cross-linked flexible sponge containing substantiallyuniformly distributed fibrous reinforcement, under such conditions thatswelling of said sponge is controlled to a water retention value of 2 to6; and, simultaneously or subsequently, reacting the resultingcross-linked sponge with a reagent which introduces functional groupsinto the modified cross-linked sponge.

The fibrous reinforcement preferably comprises cotton linters, typicallypresent in an amount of about 2 to 50% by weight; materials such asjute, cotton fibres, flax or other hydrophilic fibres may alternativelybe used. Such hydrophilic fibres preferably contain free hydroxy whichare reactive with the cross-linking agent used; the fibres arepreferably cellulosic. In some embodiments, nylon or other hydrophobicfibres may be used, in addition to or in replacement of the hydrophilicfibres. The fibrous reinforcement used according to the inventionprovides a supporting structure which permits chemical modification ofthe pre-formed sponge without complete disintegration, and also providesthe sponge with sufficient rigidity for the latter to be suitable foruse as a chromatographic adsorbent. (The sponge materials prepared asdescribed in GB 914421 and 1387265 would be too compressible to be usedas chromatographic adsorbents.)

Preferably the flexible sponge is a polymeric material which typicallycomprises any of the following: polysaccharides (such as regeneratedcellulose or cross-linked dextrans), polyvinyl alcohol, polystyrene orpolyurethane. Hydrophilic sponge polymers are preferred however, becausehydrophobic materials are known to be less suitable for chromatographicwork with proteins. This is because irreversible binding anddenaturation of the protein often occurs as a result of the use of suchhydrophobic materials. Particularly preferred hydrophilic spongepolymers are polysaccharides (such as regenerated cellulose, which canbe solubilised and then regenerated in a number of ways). Typically thiscan be done either via an intermediary product such as an ester, by theviscose process, or by dissolution in cuprammonium hydroxide.Regenerated cellulose is also particularly preferred because the ionexchange groups can be advantageously located in easily accessible sitessuch as the pore surfaces (as opposed to being buried within the body ofthe adsorbent medium) and this helps in achieving fast kinetics ofadsorption.

In the case of other polymers, such as vinyl polymers and styrenepolymers and polyurethane polymers (such as those derived frompolyisocyanates and polyethers or polyamides), it may be preferable tomix the pore forming agent with the monomers or prepolymers, so that thepolymerisation (and cross-linking) takes place in situ.

The sponge body may be further supported by a rigid mechanical supportsuch as wire or an open mesh, typically of nylon or other hydrophobicfibres, although in some embodiments, the body may contain one or morehydrophilic meshes made from such material as cotton scrim or flax.Therefore in one embodiment, the flexible cellulosic sponge may bemoulded about the mechanical support before regeneration of thecellulose. In another embodiment, the mechanical support may beintroduced into the flexible cellulosic sponge after regeneration of thecellulose and may be held in place by the use of a suitable adhesive.

The nature of the porous structure of the flexible sponge to be used ina method according to the present invention, is determined by the natureof the chromatographic separation in which it is required to be used,and can therefore be varied so as to be suitable for its required use.Typically, however, the porous structure of the sponge used in a methodaccording to the invention, has a total void volume in the range ofabout 70 to 98% (preferably 80 to 96%) of which the fractional voidageis no more than 95% thereof.

It is generally preferred that the flexible sponge contains primary andsecondary pores. The primary pores are interconnecting pores which aredimensioned so as to allow the free passage of the process liquidsthroughout the sponge. The secondary pores are provided in the walls ofthe primary pores, the former housing the majority of chromatographicadsorption sites. It is beneficial if the rate of diffusion of thechromatographic solution in and out of the pores is rapid. It istherefore preferable that the walls between the primary pores are thinand rich in suitably sized secondary pores to maximise kinetic rates.

The production of a porous structure comprising interconnecting poresgenerally involves contacting a solution of the sponge-forming polymericmaterial with a pore-forming agent such as a gas. In the case where thepolymeric material is cellulose, the sponge-forming polymeric materialis contacted with the gas either prior to, or simultaneous with,regeneration of the sponge. Either a gas, or gas forming materials,is/are introduced into the polymeric solution. Examples of gas-formingmaterials include solids, volatile liquids, chemical reagents (such ascalcium carbonate and acid), thermally decomposable materials (to causeevolution of a gas by, for example, decomposition of bicarbonate) orbiological agents (such as dextrose and yeast).

It is particularly preferred that the gas-forming materials are solidreagents such as powders, crystals, oils, waxes or ground biologicaltissue. The use of solids as precursors for the gaseous pore-formingagents is more suitable for the production of primary pores thansecondary pores; this is because it is often extremely difficult toproduce solid particles of a sufficiently small size generally requiredfor the production of secondary pores. In the case where the polymericmaterial is cellulose the reagent may be removed either after or duringthe regeneration of the cellulose into solid form, the removal typicallyinvolving either treatment with an acid, an alkali, or an enzyme, theuse of electromagnetic energy or solvent action.

A particularly preferred method of producing a sponge having aninterconnecting porous structure involves use of a xanthate; the lattermethod is preferred because the size of pores produced by gas given offby the xanthate can be varied by varying the degree of substitution ofthe latter.

A further preferred method of producing a sponge having aninterconnecting porous structure involves the use of crystals ofhydrated sodium sulphate where particles of varying sizes can again beused. For a high resolution on a laboratory scale, crystals of hydratedsodium sulphate having a particle size in the range from 200 to 400microns can be used to make the interconnecting pores. For a largerscale commercial separation requiring high flow rates, crystals ofhydrated sodium sulphate having a particle size of 1500 to 3000 micronscan be used. It is particularly difficult to use hydrated sodiumsulphate with a particle size less than 100 microns to formcorrespondingly sized pores in the sponge. However, when it is requiredto produce a sponge having a pore size in the order of 100 microns othersolids such as calcium carbonate may be used.

The actual volume of the primary pores (i.e. fractional voidage) isdependent on the amount of primary-pore forming agent introduced intothe sponge-forming polymeric solution and as hereinbefore described cantake any value up to 95% of the total void volume.

The thickness of the walls of the primary pores largely depends on thequantity of primary pore forming agent which is mixed with the solutionof sponge forming polymeric material. Among other factors, the minimumwall thickness will depend on how closely the particles of the primarypore forming agent fit together. The overall amount of functional groupswhich can be introduced per unit volume into the flexible sponge willgenerally be increased as the density of the sponge is increased. Thethickness of the primary pore walls may be varied depending on therequired use of the sponge adsorbent medium. For example, if the mediumis used for the processing of mineral ions, then a thick wall can beused because the diffusion rates for mineral ions are fast. However, ifthe medium is used for the chromatography of macromolecules such asproteins, then a thin wall would be preferred because of the slow rateof diffusion of the macromolecules through the wall. It is not possiblehowever to obtain uniform wall thicknesses within a sponge medium (e.g.a cellulosic sponge medium is known to have a wall thickness rangingbetween 5 and 45 microns). In a method according to the presentinvention, the resultant adsorbent medium generally comprises aninterconnecting porous structure where the primary pore wall thicknessis typically in the range of about 2 to 300 microns.

In the use of the porous sponge media, it is desirable to achieve plugflow (which is associated with the best quality separations) and tominimise axial dispersion or back mixing. A narrow range of primarypores sizes is therefore preferred, and overlarge pores which can beassociated with non uniform flow should be eliminated.

The secondary pores may be naturally occurring in the polymer as aresult of variation in its density or may be formed by the use of a poreforming agent or agents which are generally used in conjunction with theprimary pore forming agent. Typically the secondary pores are smallerthan the primary pores and may be formed by any of the followingmethods.

A solution of the polymeric material may be mixed with a removablereagent having individual particles of a predetermined size, so as toproduce a flexible sponge having a desired secondary pore structure(i.e. a structure having a pore size determined by the reagent particlesize). A liquid (probably immiscible with the polymer solution) can beadded to the polymer solution which upon mixing forms continual channelswithin the liquid polymer. Alternatively, the density of the polymersolution (in this case cellulose) may be lowered by the addition of asuitable solvent so that when the cellulose is regenerated the resultingsponge has an open pore structure. Suitable methods of removing theadded gas or liquid are as above.

The secondary pores may of course be produced by contacting the polymersolution with a solid pore-forming agent, however this method isgenerally less successful in producing secondary pores of a requiredsize (as previously described).

The resulting medium may have higher flow rates than conventionaladsorbent media. For example, when used in a column of height 4 mm andan internal diameter of 43 mm, under a pressure of one bar, flow ratesin excess of 50 meters per hour may be achieved (for example about 90meters per hour). This compares with good commercially available media(such as those commercially available under the trade names Whatman CM52and Indion HC2, both of which are carboxymethyl celluloses), where undersimilar conditions, flow rates of less than 40 meters per hour areachievable.

In the method according to the present invention, cross-linking isparticularly important in maintaining the predetermined porous structureand also to protect the porous sponge and derivatives thereof againstdeterioration due to chemical and physical attack. It is preferred thatcross-linking of the sponge comprises contacting the sponge with aliquid having dissolved therein a chemical cross-linking agent for thesponge. In the case of a cellulose sponge the cross-linking agent can beadded either during or after regeneration of the cellulose sponge andmay be added in one or more stages. A preferred embodiment howeverinvolves the addition of the cross-linking agent to the solution ofsponge forming cellulosic material during the regeneration process. Inthis way the pores are substantially maintained at their predeterminedsize. Alternatively cross-linking is carried out after regeneration iscomplete. In this case the porous cellulose sponge is first swollen togive the desired pore size, and then cross-linked to hold the porousstructure.

The liquid having the chemical cross-linking agent dissolved therein maybe an aqueous alkaline solution, generally comprising sodium hydroxide.Such an aqueous alkaline solution typically contains sodium hydroxide inan amount of 0.5 to 7 molar, preferably slowly raised from 0.7 to 5molar over the period of one hour. Alternatively a unimolar solution canbe used over the same time period. If the molarity of the solution iseither too high or too low, the flow rate of liquid through theresultant adsorption medium and the mass-transfer kinetics are impaired.If too high, undesirable gel formation may result, with deleteriouseffect on the flow rate.

The nature of the cross-linking agent will depend on the flexible spongematerial. In the case of polysaccharides such as cellulose a polarcross-linking agent may be used which is soluble in aqueous media.Examples of suitable such cross-linking agents include formaldehyde,dichlorhydrin, epichlorhydrin, dibromomethane, bis-epoxypropyl ether, a1,4 butane diol bisepoxy ether, dialdehydes such as glyoxal, and divinylcompounds such as divinylsulphone. Preferred cross-linking agents aredichlorhydrin (which is most preferred) and epichlorhydrin.

The cross-linked sponge may be further treated with, for example hotsodium hydroxide solution. This will solubilise parts of the sponge(particularly low molecular weight fractions) which have not beencross-linked and therefore open up the sponge structure. However, thereaction conditions are chosen according to the nature of cross-linkingagent, for example epoxy compounds such as epichlorohydrin arepreferably used in an alkaline medium, whereas aldehydes such asformaldehyde are preferably used in an acidic medium.

In the case where the sponge is formed in situ then the cross-linkingagent may be part of the sponge forming process (such as the use ofdivinyl benzene to cross-link polystyrene).

It is recognised that the nature of the flexible sponge, including thegeneral porous structure, may depend on a wide range of factors whichinfluence the manufacturing system. For example changes may be made tothe concentration, degree of polymerisation, or viscosity of thepolymer. Agents (such as surfactants) likely to effect the secondary andprimary pore structure may be added.

The reagent which is subsequently reacted with the cross-linked spongemay be one which introduces ion-exchange groups. Examples of suchreagents are compounds containing amino, alkylamino or quaternaryammonium groups (when it is desired to produce an anion exchange resin),or compounds containing sulpho, phospho or carboxyl groups (when it isdesired to produce a cation exchange resin).

Examples of the former type of compound are diethylaminoethyl chloride(optionally followed by reaction with bromoethane to produce thecorresponding quaternary derivative), chlorohydoxypropyl trimethylammonium chloride, glycidyltrimethylammonium chloride,polyethyleneimine, di-(hydroxyethyl)-aminoethyl chloride andp-morpholino ethyl chloride; examples of the latter type of compound arechloroacetic acid, chlorohydroxypropane sulphonic acid, sodiumbisulphate, bromoethane sulphonic acid, hydroxyethane sulphonic acid,chloroethane sulphonic acid, chlorosulphonic acid, chloromethanesulphonic acid and 1,3-propane sultone.

Other types of functional groups which may be introduced by the reagentwhich is reacted with the cross-linked sponge include metal chelates,antibodies (such as IgG), antigens (such as Protein A), dyes, lectins,or groups which can fix biologically active materials such as enzymes.An example of the latter type of group results from the reaction of thesponge successively with carbonyl diimidazole, p-amino-benzamide andhexanoic acid; an example of the introduction of chelate groups is bysuccessive reaction with a diglycidyl ether, sodium borohydride (toproduce epoxy groups), iminodiacetic acid, and a zinc or copper salt.

The resulting sponge adsorbent medium can be further treated so as tomodify the pore surface chemistry. For example the pore surface can becoated with a materials such as polyethylene imine or DEAE dextran, orchemically grafted to produce polymers such as polyacrylic acid,polyethylene imine or materials such as phosphatidyl choline derivativesso that they occupy some part of the space inside the pore.

GB 1387265 describes the production of an ion exchange sponge byintroducing ion-exchange groups before regeneration of the cellulose.The method according to the present invention allows such ion exchangegroups to be introduced into regenerated cellulose sponge. According tothe present invention the ion-exchange groups can be introduced into theregenerated cellulose sponge by spraying or soaking of the cellulosesponge with a suitable reagent. Any excess reagent can be removed by theaction of a roller, under vacuum or by heating. The latter may beeffected by convective or radiant heat processes such as curing, ormicrowave or radio frequency radiation. Alternatively the cellulosesponge may be placed in a housing and the reactive solutions passedthrough the sponge. Heating may be affected by the use of a pre-heatedinert liquid or gas.

The body of flexible sponge material used in accordance with the presentinvention may be in the form of a block, an annulus, a continuous sheet,a rolled sheet, a disc, a tape, a rod, a pad or the like. Although it ispossible to use the adsorbent medium in a free form, it is generallyused in apparatus having an inlet for liquid, an outlet for liquid(which may in some embodiments, be the same as the inlet or, in otherembodiments spaced from said inlet) so as to define a path for liquidflow between the inlet and the outlet, said adsorbent medium beingpositioned across said liquid flow path.

The adsorbent medium is generally used in block form in the apparatuswhich means that there is the possibility that liquid could flow betweenthe internal wall of the apparatus and the adsorbent medium. Thisproblem may be overcome in a variety of ways such as the use of asealant or an adhesive or both. A preferred embodiment however is to usethe adsorbent medium under compression across the liquid flow path suchthat liquid flows from the inlet through the adsorbent medium.

The degree of compression is preferably such that the lateral dimensionis at least 1%, more preferably at least 3% (such as 3 to 10%) less thanthe corresponding dimension of the adsorbent medium in unrestrainedform.

The use of the adsorbent medium under lateral compression in theapparatus according to the invention is such that short-circuiting (thatis, passage of liquid through the apparatus without contact with theadsorbent medium) is avoided.

In one embodiment of the present invention, the adsorbent medium may bein the form of a flexible block typically in the form of a cylinder withthe inlet and/or outlet connected to the axial core to the cylinder. Itis preferred that the adsorbent medium should be under substantiallyuniform compression throughout its volume.

The apparatus may be primed for use by inserting the adsorbent medium inthe liquid path in at least partially dehydrated form, and allowing theadsorbent medium to undergo hydrophilic swelling such that it islaterally compressed by the walls of the liquid path. Alternatively, theadsorbent medium, in hydrophilic swollen state, may be preliminarilycompressed prior to insertion in the liquid path followed by partialrelease of the compressive restraint when the adsorbent medium has beeninserted in the liquid path. In a further alternative, the adsorbentmedium may be compressed after insertion into a liquid path bycompressive means acting on one or more of the walls defining the liquidpath.

When the adsorbent medium is in the form of discs or pads, a pluralityof such discs or pads can be stacked in a column for use, for example inchromatographic separation, when material to be separated could beselectively eluted from different pads or discs, or parts thereof, inthe stack.

In one embodiment of the invention in which a stack of such discs orpads are employed, it may be advantageous to provide indicator means toindicate different levels in the stack. For example, different colourscan be employed to indicate different levels in the stack; discs fromdifferent levels may be removed from the stack and treated separatelyfor isolation of respectively fractionated material therefrom.

The apparatus and adsorbent medium according to the invention may beused in any conventional adsorption process, such as the isolation ofproteins (for example, from dairy or soya whey), or mineral ions,adsorption of polyelectrolytes (e.g. humic acid), or radioactivereagents by ion-exchange from liquid phase material, affinitychromatography, or immobilisation of enzymes, or lysozyme separation.

The apparatus according to the invention may be provided with means forcompression of the adsorbent medium, whereby the latter can besuccessively compressed and decompressed, for example, following anadsorption phase and/or following a washing phase. Material adsorbed bythe ion-exchange material can be adsorbed either in the compressed stateor in the decompressed state (in the latter case the desorption phaseis--preferably followed by successive compression and decompression).When desorption takes place with the adsorption medium in the compressedstate, this may involve an adsorption phase with a relatively high voidpercentage, followed by a desorption phase with a relatively low voidpercentage.

The use of successive compression/decompression phases enables a highdegree of liquid-solid contact to be obtained in the adsorption phase,followed by efficient desorption of treated liquid; the yield ofmaterials such as polyelectrolytes can be thereby increased, and thevolume of liquid product can be decreased (that is, its concentrationcan be greater).

For some purposes, the adsorbent medium may be used partially compressedthroughout, with remarkably little change in flow characteristicscompared to the uncompressed medium. This enables improved volumetricefficiency to be obtained (that is, more adsorbent medium can beemployed per unit volume, without substantial impairment of liquidflow).

According to another aspect of the invention, therefore, there isprovided a method of isolating material from a liquid phase whichcomprises flowing liquid from the inlet to the outlet of apparatusaccording to the invention so as to cause said material (which istypically a polyelectrolyte material, a protein or the like) to beadsorbed from the liquid, terminating the flow of liquid, andcompressing the adsorbent medium. This compression allows elution with alower volume of desorbent.

The adsorbent medium need not necessarily be used vertically; forexample, in some embodiments, it may be used horizontally (unlike, say,conventional granular ion-exchange media, which must be used in verticalorientation).

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a graph showing comparative flow rates obtainable using acellulosic medium produced according to the present invention incomparison with other cellulosic media.

FIG. 2 is a graph showing comparative velocities through a cellulosicmedium according to the present invention in comparison with othercellulosic media.

FIG. 3 is a graph showing a comparison of the adsorption kinetics ofregenerated cellulosic sponge according to the present invention incomparison with the adsorption kinetics of other cellulosic media.

FIG. 4 is a graph showing the effect on bed resistance when a spongeaccording to the present invention is compressed by differing factors.

FIG. 5 is a graph showing the resulting loading and elution profileaccording to Example 6 of the specification.

FIG. 6 is a graph showing the results of a comparison drawn between twocarboxymethyl derivatives according to Example 7 of the specification.

According to the invention the sponge adsorbent medium may be reduced toa powder which may then be reconstituted into a solid form, e.g. by theuse of an adhesive.

The invention will now be illustrated by reference to the followingExamples and Figures, which do not limit the scope of the invention inany way.

Examples 2,3, and 4 illustrate methods of treating a cross-linkedcellulose sponge obtained by the method of Example 1, with a reagentwhich introduces functional groups. Examples 5 to 12 illustrate theproperties of a resultant treated sponge.

EXAMPLE 1 a). Production of Viscose

500 g of alkaline cellulose was prepared containing 137.5 g ofcellulose, 77.5 g of NaOH and 285 g of water. The average degree ofpolymerisation of the cellulose had been reduced to approximately 200 byalkaline oxidative degradation. This was transferred to a z-arm mixerand reacted with 90 g of carbon disulphide at 32° C. for 60 minutes. 250g of 19% NaOH and 1275 g of chilled water were then added to makeviscose.

The viscose was then further processed according to any of the followingmethods to produce a cellulose sponge from which anion and cationexchangers or affinity material could be produced. In each case a flatsheet (although other forms such as a block or annulus could beproduced) of porous adsorbent material with a thickness of approximately5 mm was produced, which was washed and treated with 0.1M HCl todissolve the calcium carbonate, and then further washed and de-watered.In method 2, 50 g of the porous sheet material was further treated withdichloropropanol.

Method 1

To 1500 g of the viscose was added: 32 g of cotton linters and 5000 g ofcrystals of sodium sulphate decahydrate (particle size range from 1000to 3000 microns). The resulting mixture was blended to produce a paste.The paste was then molded between 2 plates of perforated stainless steeland regenerated in sodium sulphate solution at 95° C.

Method 2

To 1500 g of the viscose was added: 32 g of cotton linters, 3900 g ofcrystals of sodium sulphate decahydrate (particle size range from 200 to400 microns), and 1000 g of powdered calcium carbonate (particle sizeless than 2 microns). The resulting mixture was blended to produce apaste. The paste was then molded between 2 plates of perforatedstainless steel and regenerated in sodium sulphate solution at 95° C.

b). Production of Cross-Linked Cellulose Sponge

Method 3

To 1500 g of the viscose was added: 15 ml of epichlorohydrin, 80 g ofcotton linters, 5000 g of crystals of sodium sulphate decahydrate(particle size range from 1000 to 3000 microns), and 1000 g of powderedcalcium carbonate (particle size less than 2 microns). The resultingmixture was blended to produce a paste. The paste was then moldedbetween 2 plates of perforated stainless steel and regenerated in sodiumsulphate solution at 95° C. The resulting regenerated material had awater retention value of 3.2 and a porous volume of 91%. In this methodcross-linking was achieved simultaneous with regeneration of the sponge.

Method 4

50 g of cellulose sponge produced according to method 1 was treated with0.6 to 5.0M NaOH over a one hour period with 250 to 2000 ml of liquidcontaining 1% to 5% v/v of dichlorohydrin. The sponge was subsequentlycured at 60° C. for up to 1 hr. The resulting material was a porouscross-linked cellulose sponge from which anion and cation exchangers oraffinity material may be produced. The resulting material had a waterretention value of 3.4; similar runs can be operated with waterretention values in the range 2 to 6. The porous volume of the resultingmaterial was determined by column tracer flow using acetone, and wasfound to be 92%; similar runs can be operated to give porous volumes inthe range 70 to 98%. A sample of the material was cut to obtain a cleancross-section and freeze dried to remove water. The latter was thenexamined by SEM. The average primary wall thickness was estimated to beabout 20 microns; similar runs can be operated to give average wallthicknesses in the range 2 to 300 microns. The variance in wallthickness could be achieved by changing either the cellulose content ofthe viscose or the amount of cotton linters and pore forming agent inthe paste.

Method 5

50 g of cellulose sponge produced according to Method 2 was subjected tocross-linking as in Method 4. The resulting material had a waterretention value of 3.3%, a porous volume of 94% and a primary wallthickness of 7 microns.

EXAMPLE 2

Carboxymethyl cellulose was produced by taking 50 g of a cross-linkedcellulose sponge, obtained by method 4 of Example 1, and adding thereto400 ml of a solution of 5M NaOH and 80 g of sodium chloroacetate andmaintaining the mixture at 100° C. for 1 hr. The resultant medium has aprotein capacity of 2 g per dry gram of sponge with a maximum liquidflow in excess of 40 meters per hour and also a water retention value of3.2.

EXAMPLE 3

Sulphopropyl cellulosic sponge is produced by taking 50 g of across-linked cellulose sponge, obtained by method 4 of Example 1 andadding thereto 1000 ml of a solution containing 5M NaOH and 590 g of thesodium salt of chlorohydroxy propane sulphonic acid and maintaining themixture at a temperature of 100° C. for 3 hrs. The resultant cellulosicsponge material has a protein capacity of one gram per dry gram with amaximum liquid flow rate in excess of 40 meters per hour and a waterretention value of 3.4.

EXAMPLE 4

Quaternary methyl ammonium cellulosic sponge is made by taking 50 g of acellulose sponge, obtained by method 4 of Example 1 and adding thereto900 ml of liquid containing 5M NaOH, 164 g of chlorohydroxy propyltrimethyl ammonium chloride and 1.74 g of sodium borohydride andmaintaining the mixture at 50° C. for 2 hrs. The resultant cellulosicsponge material has a protein capacity of 1.5 g per dry gram with amaximum liquid flow rate in excess of 40 meters per hour and a waterretention value of 3.3.

EXAMPLE 5

FIG. 1 shows comparative flow rates through three cellulosic media.Curve A corresponds to a medium obtained according to Example 4, Curve Bcorresponds to CM52 and Curve C corresponds to Indion HC2. The flowrates were measured using a column of height 4 mm and an internaldiameter of 43 mm, under a pressure of one bar. As seen in FIG. 1, aflow rate of about 90 meters per hour is obtainable using a cellulosicmedium produced according to the present invention, whereas flow ratesof less than 40 meters per hour (about 20 and 35 respectively) wereobtained using CM52 and Indion HC2.

FIG. 2 snows comparative velocities through cellulosic media. A columnof 100 mm×26 mm was packed with a cellulosic medium obtained accordingto Example 4 and a maximum flow rate therethrough of 10 meters per hourwas achieved (Curve A), the column was similarly packed with CM52 andHC2 both achieving maximum flow rates of less than 1 meter per hour(Curves B and C respectively).

A column of 100 mm×147 mm was also packed with a cellulosic mediumobtained according to Example 4 and a maximum flow rate of 9 meters perhour was obtained (Curve D).

Referring to FIG. 3, there is shown a comparison of the adsorptionkinetics of regenerated cellulosic sponge obtained according to Example4 with the adsorption kinetics of HC2 and CM52 cellulosic media. Allthree media were arranged in a 30 mm×25 mm via column adsorbing proteinin recirculation batch mode at a flow rate of 4 meters per hour. Thedimensionless rates for the three media were 0.35, 0.15 and 0.1respectively.

FIG. 4 shows the effect on bed resistance when sponge made according tothe invention is compressed by a factor of 1.25 (Curve A) and a factorof 2 (Curve B).

EXAMPLE 6

Cross-linked porous regenerated cellulose was made according to Example1 and converted to carboxymethyl cellulose according to Example 2. Thiswas then used for the separation of ovalbumin, conalbumin and lysozymefrom fresh egg white, in a single step. The conalbumin and lysozyme wereassumed to be 95% pure by gel electrophoresis and the overall processhad a productivity in excess of 80 kg/m/hr.

130 ml of 14 mg/ml of fresh egg white of pH 4.8 and ionic strength of2.3 ms/cm is fed to a bed of the CM-porous sponge media contained in acolumn of 250×10 mm diameter and recycled 18 times through the column.After washing the column with loading buffer of 0.01M sodium acetate atpH. 4.8 for 3 minutes, elution of the conalbumin and lysozyme waseffected with a salt gradient using 250 ml of 0.6M sodium chloridesolution and 250 ml of 0.01M sodium acetate buffer at pH 4.8 at a flowrate greater than 9 meters/hr. The resulting loading and elution profileare illustrated in FIG. 5.

This example shows that high resolution separations can be achieved atfast flow rates using a sponge adsorbent medium with primary pores whichmake it suitable for commercial scale operation.

EXAMPLE 7

In this Example a comparison is drawn between two carboxymethylderivatives prepared as in Example 2, the first derivative beingprepared from viscose having primary pores in the range 1500 to 3000microns (as produced according to Method 1 of Example 1) and the secondderivative being prepared from viscose having primary pores in the range250 to 500 microns respectively (as produced according to Method 3 ofExample 1). The results of the experiment are shown in FIG. 6.

In both cases the CM-Porous sponge media was packed into a column of20×25 mm diameter and a 0.01M sodium acetate buffer solution, at pH 4.5,containing 1 mg/ml of lysozyme, was pumped through the columns at a flowrate of 4 meters per hour until the lysozyme concentrate in the outputwas 50% of that at the input of the column. The quality of resolutionwas similar in both cases. The time required to reach the breakthroughpoint was three times longer for the small pore structure compared tothat for the larger pore structure. This is considered to be because ofgreater capture efficiency and higher density. In both cases a high flowrate which was considered to be superior to that which could be obtainedwith particulate material was maintained. To illustrate the amount ofbound lysozyme the latter was eluted from the column in a single stepwith carbonate buffer at pH 9.

EXAMPLE 8

This example demonstrates the use of compression of a porous adsorbentmedium to concentrate a dilute protein solution during an adsorptionprocess. A 320 times concentration factor of the protein solution wasachieved.

Cross-linked porous cellulose sponge was prepared according to Method 4of Example 1. The sponge had a porous structure with primary pores inthe range of 1000 to 3000 microns and was converted to make CM celluloseporous sponge according to the method of Example 2. The sponge was thencut into a disc of dimensions 5 mm×25 mm, diameter and inserted into asmall column. 1500 ml of lysozyme at a concentration of 0.1 mg/ml in 50mM acetate buffer at pH 4.5 was then recirculated through the disc at aflow rate of 36 m/hr for 30 minutes. The disc was then washed with atleast 10 times the disc volume of loading buffer and mechanicallysqueezed to remove excess buffer. 2.5 ml of 0.25 mM sodium carbonate, pH10.6 buffer was added to saturate the disc. After five minutes theelution buffer was mechanically squeezed from the disc and collected;the buffer contained lysozyme at a concentration of 32 mg/ml.

EXAMPLE 9

This example shows that an adsorbent medium produced by a methodaccording to the present invention comprises channels having a greaterproportion by volume of the medium than do the corresponding channels ofa particulate adsorbent material. This is an important reason why anadsorbent medium produced by a method according to the present inventionhas superior flow characteristics compared to known particulatematerials.

A comparison is drawn between the total pore volume and the voidage (theprimary pore volume/the space between particles) for CM cellulosesponge, Pharmacia Fastflow Sepharose CM, Whatman CM52 and High capacityPhoenix CM. A column of height 50 mm and diameter 10 mm containing therespective adsorbent material was set up in each case and size exclusiondata determined using 0.5% acetone and 0.9 g of blue dextran (molecularweight at 2,000,000). 0.25 ml of each of these solutions was injectedinto the stream of a column flowing with water at a flow rate of 0.35ml/min. The values for acetone show the total void volume and the valuesfor blue dextran the fractional voidage. The results are shown in thefollowing table.

    ______________________________________                                                          Acetone Blue Dextran                                        Porous Vol.       %       %                                                   ______________________________________                                        Sponge Adsorbent  94      69                                                  Medium (produced by a                                                         method according to                                                           the present invention)                                                        Pharmacia         94.5    43                                                  Whatman           82      25                                                  Phoenix           92.5    23                                                  ______________________________________                                    

EXAMPLE 10

This example demonstrates the superior flow rate and kinetic propertiesof a sponge adsorbent medium according to the present invention ascompared to those properties of a particulate medium. A single protein,Human Serum Albumin (HSA), is studied for simplicity. The maximumcapacities (Qm) and dissociation constants (Kd) were determined forPharmacia Fastflow DE Sepharose and quaternary methyl ammonium (QMA)sponge as prepared in Example 4. The results were: for the PharmaciaSepharose, Qm=98 g/l and Kd=0.2 mg/ml; for the sponge Qm=19 g/l andKd=0.04 mg/ml.

In most large scale commercial processes the product of interest isoften present at a concentration of approximately 1 mg/ml. In order totest the relative potential productivities a solution of HSA at 1.0 g/lwas loaded onto columns of similar capacity containing PharmaciaSepharose and the sponge adsorbent medium respectively. The columncontaining the Pharmacia Sepharose was loaded and eluted at a flow rateof 0.3 m/hr, with 1 g/l of the HSA in 0.05M Tris based buffer at pH 7.5and a 0.05M sodium acetate buffer at pH 4.5 respectively. A potentialproductivity of 19 Kg/m³ /hr at yields of 84% and an elutedconcentration of 5.2 g/l was achieved.

A similar experiment was carried out using the adsorbent sponge exceptthat the column was loaded using feed recirculation at a velocity of 9.2m/hr. A potential productivity of 40 kg.m³ /hr at a yield of 95% and aneluted concentration of 4.8 g was achieved.

EXAMPLE 11

In order to illustrate the effect of the fibrous reinforcement, samplesof sponge with and without fibrous reinforcement were made according toExample 1. The compressive modulus was then measured in a fullysaturated condition after immersing the sponge in distilled water for 48hours. The compressive modulus for the non reinforced material was 0.2MPa whereas that for the reinforced material was 2 MPa. The nonreinforced sponge was considered too compressible for its proposed useand deteriorated easily during attempts to further process it.

EXAMPLE 12

This example shows how the properties (e.g. total void volume,fractional voidage and primary pore wall thickness) of a regeneratedcellulose sponge are dependent on the components present in the solutionof sponge forming material from which the cellulosic sponge isregenerated.

Four viscose samples were produced according to Example 1, thecomposition of each viscose sample is shown in the following table. Eachviscose sample was regenerated and cross-linked according to methods 1and 3 of Example 1 respectively. The quantity of hydrated sodiumsulphate and cotton linters used in the regeneration of the viscose wasvaried for each viscose sample.

The following table illustrates the individual compositions andproperties of each viscose sample.

    ______________________________________                                        Sample         1      2         3    4                                        ______________________________________                                        Components present                                                            in the solution                                                               from which the                                                                cellulose sponge                                                              is regenerated                                                                Cellulose %    1.5    1.5       3.0  3.0                                      Cotton Linters %                                                                             0.5    0.5       1.0  1.0                                      Sodium Sulphate %                                                                            76     60        76   60                                       Total Void Volume %                                                                          94     91        86   83                                       Fractional Voidage %                                                                         69     51        65   50                                       Wall Thickness 20     47        NT   NT                                       (microns)                                                                     ______________________________________                                    

The main effect of lowering the quantity of hydrated sodium sulphatepresent in the polymeric solution is to reduce the voidage of theresultant cellulose sponge medium.

Increasing the quantities of cellulose and fibrous reinforcement in thepolymeric solution decreases the total porous volume of the cellulosesponge adsorbent medium. Sample 1 was considered suitable for theadsorption of macromolecules. Sample 4 however could be substituted togive the highest ion exchange capacity per unit volume and was mostsuitable for the adsorption of small mineral ions.

What is claimed is:
 1. A method of producing a cross-linked cellulosesponge, comprising:a) providing:i) viscose, ii) at least onecross-linking agent selected from the group consisting ofepichlorohydrin, dichlorohydrin, formaldehyde, dibromomethane,bis-epoxypropyl ether, 1,4 butane-diol-bisepoxy ether, epoxies,dialdehydes, glyoxal, divinyls, divinyl benzene, and divinylsulphone,iii) at least one pore-forming agent, and iv) a molding means; b) mixingsaid viscose, said cross-linking agent, and said pore-forming agent toproduce a paste; and c) molding said paste with said moulding means,under conditions such that a cross-linked cellulose sponge is produced.2. The method of claim 1, wherein said pore-forming agent is selectedfrom the group consisting of sodium sulfate, calcium carbonate, andxanthate.
 3. The method of claim 2, wherein said sodium sulfate issodium sulfate decahydrate.
 4. The method of claim 1, further comprisingthe step d) introducing at least one functional group into saidcross-linked cellulose sponge.
 5. The method of claim 2, wherein saidfunctional group is selected from the group consisting of ion-exchangegroups, metal chelates, antibodies, antigens, dyes, lectins, andenzymes.
 6. A method of producing a cross-linked cellulose sponge,comprising:a) providing:i) viscose, ii) at least one cross-linking agentselected from the group consisting of epichlorohydrin, dichlorohydrin,formaldehyde, dibromomethane, bis-epoxypropyl ether, 1,4butane-diol-bisepoxy ether, epoxies, dialdehyde, glyoxal, divinyls,divinyl benzene, and divinylsulphone, iii) sodium sulfate and iv) amolding means; b) mixing said viscose, and said sodium sulfate toproduce a paste; c) molding said paste with said moulding means, underconditions such that a cellulose sponge paste is produced; and d)exposing said cellulose sponge paste to said cross-linking agent, underconditions such that a cross-linked cellulose sponge is produced.
 7. Themethod of claim 6, further comprising the step d) introducing at leastone functional group into said cross-linked cellulose sponge.
 8. Themethod of claim 6, wherein said functional group is selected from thegroup consisting of ion-exchange groups, metal chelates, antibodies,antigens, dyes, lectins, and enzymes.
 9. A method of producing across-linked cellulose sponge having functional groups, comprising:a)providing:i) viscose, ii) at least one cross-linking agent selected fromthe group consisting of epichlorohydrin, dichlorohydrin, formaldehyde,dibromomethane, bis-epoxypropyl ether, 1,4 butane-diol-bisepoxy ether,epoxies, dialdehyde, glyoxal, divinyls, divinyl benzene, anddivinylsulphone, iii) means for creating primary pores having walls of apredetermined thickness, iv) at least one functional group, and iv) amolding means; b) mixing said viscose, said cross-linking agent, andsaid means for creating primary pores, to produce a paste; c) moldingsaid paste with said moulding means, under conditions such that across-linked cellulose sponge is produced; and d) introducing said atleast one functional group into said cross-linked cellulose sponge. 10.The method of claim 9, wherein said functional group is selected fromthe group consisting of ion-exchange groups, metal chelates, antibodies,antigens, dyes, lectins, and enzymes.
 11. The method of claim 9, whereinsaid pore-forming means is selected from the group consisting ofhydrated sodium sulfate, calcium carbonate, and xanthate.
 12. The methodof claim 11, wherein said hydrated sodium sulfate is sodium sulfatedecahydrate.
 13. The method of claim 9, wherein the wall thickness ofsaid primary pores is about two to three hundred microns.